A simple, rapid fluorometric assay for the determination of glucose 6-phosphate dehydrogenase activity in dried blood spot specimens.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy of human beings and is the most common cause of jaundice and acute hemolytic anemia in South East Asia. The deficiency causes acute hemolytic anemia following ingestion of 6-amino quinoline antimalarials, phenacetin, and other substances. The rapid identification of infants or patients with this deficiency would help to prevent their exposure to these substances and subsequent risk to health. The assay is relatively simple. A 3mm punch from a dried blood spot sample is placed in a well of a black fluorescent microtiter plate containing calibrators and controls in duplicate. 100 microl of reagent is added and the sample is allowed to react for 30 minutes at ambient temperature after which 200 microl of stop reagent is added. The plate may be read immediately or up to one hour in a fluorescent reader (ex 355 nm: em 460 nm). Glutathione. ascorbate and bilirubin do not affect the assay. hemoglobin does quench the fluorescence by about 1.1 fluorescence units/g/dHb. This would not cause any false negatives and deficients would not be missed. G6PD activity in whole blood normal samples was examined at -20, 6 and 37 degrees C over 14 days. The samples lost about 20% activity after 48 hours and 31% by the end of 14 days. The samples stored at -20 degrees C and 6 degrees C remained relatively stable over this period. In a preliminary study eight diagnosed G6PD deficient samples had a mean value of 2.0 U/gHb (range 0.8 to 4.4) and fell within 3 SD units of the mean. Forty one normal samples had a mean of 6.6 micromol/min/gHb. Only one sample with a low hemoglobin level fell outside of 3 SD units of the mean. The Wallac assay was compared to the Sigma G6PD assay and although the values appeared lower at normal levels, the deficient samples compared well.

Isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut- and bronchus-associated lymphoid tissues of suckling pigs.

Antibody-secreting cells (ASC) were enumerated in gut- and bronchus-associated lymphoid tissues of pigs exposed to three antigenically related coronaviruses: virulent transmissible gastroenteritis virus (TGEV), attenuated TGEV, and porcine respiratory coronavirus (PRCV). Exposure of 11-day-old pigs to virulent TGEV resulted in severe gastroenteritis and virus shedding mainly in feces but also to a limited extent in nasal secretions. PRCV and attenuated TGEV exposure produced no clinical signs and only one pig given a high dose of attenuated TGEV shed virus in feces, but virus was shed from the nasal passages. Nasal virus titers were highest after PRCV inoculation of pigs. Mononuclear cells were isolated from spleens, mesenteric, and bronchial lymph nodes of pigs and assayed for virus-specific IgG and IgA antibody secretion by an enzyme-linked immunospot assay. Virus-specific ASC peaked at postinoculation days 12 to 24 and IgG-ASC outnumbered IgA-ASC in all tissues tested. The greatest numbers of ASC were in mesenteric lymph nodes of virulent TGEV-exposed pigs and in BLN of PRCV-exposed pigs. Attenuated TGEV induced intermediate ASC responses in the gut and respiratory tract. Secondary in vitro ASC responses to inactivated TGEV or PRCV paralleled the primary responses except in BLN where the numbers of memory ASC were high for both TGEV- and PRCV-exposed pigs. We conclude that: 1) a single exposure of pigs to PRCV either oral-nasally or by aerosol leads to potent systemic and bronchus-associated, but not gut-associated, ASC responses; 2) a high dose of attenuated TGEV (4 x 10(8) plaque-forming units) is more effective than PRCV (6 x 10(5) or 2 x 10(8) plaque-forming units) or a lower dose of attenuated TGEV (7 x 10(6) plaque-forming units) in eliciting gut-associated ASC; 3) although virulent and a high dose of attenuated TGEV induce high numbers of ASC in the tissues tested, virulent TGEV induces the most ASC in the gut and IgA-ASC in all lymphoid tissues; and 4) virus replication in the gut or respiratory tract is a major factor affecting the magnitude of an ASC response at that site and may be necessary for the recruitment of IgG- and IgA-ASC and memory cells in large numbers from other mucosal inductive sites. This unique model of mucosal immunity using antigenically related viruses with distinct tissue tropisms may help to clarify interactions of the various components of the common mucosal immune system.

Identification of antigenic sites mediating antibody-dependent enhancement of feline infectious peritonitis virus infectivity.

We have previously demonstrated antibody-dependent enhancement of feline infectious peritonitis virus (FIPV) infection of macrophages using both virus-specific antisera and monoclonal antibodies (MAbs) to the spike (S) protein of FIPV. To increase our understanding of this phenomenon, six representative MAbs from a previously documented group of 12 enhancing MAbs were used to identify epitopes that mediate antibody-dependent enhancement of FIPV infectivity. Analysis of the results of kinetics-based competitive ELISA (K-cELISA) among these six enhancing MAbs grouped the epitopes into two clusters. Because transmissible gastroenteritis virus (TGEV) and FIPV are so closely related antigenically, we also conducted K-cELISA experiments between the FIPV MAbs and TGEV S protein-specific MAbs for which the epitopes had previously been mapped to specific sites on the TGEV S protein. Results of these assays indicated that the two FIPV epitope clusters are homologues of the previously defined TGEV S protein sites A and E/F. In addition, two TGEV S protein-specific MAbs also induced antibody-dependent enhancement of FIPV infection of macrophages. This functional cross-reactivity provides further support for the close antigenic relationship between FIPV and TGEV. Our results provide a preliminary localization of several enhancing epitopes within the amino acid sequence of the FIPV S protein.

Competition ELISA, using monoclonal antibodies to the transmissible gastroenteritis virus (TGEV) S protein, for serologic differentiation of pigs infected with TGEV or porcine respiratory coronavirus.

Monoclonal antibodies (MAB) to subsite A (25C9) and subsite D (44C11) of the S protein of transmissible gastroenteritis virus (TGEV) were used in a blocking ELISA on fixed TGEV-infected swine testis cells to differentiate sera from pigs experimentally inoculated with either TGEV or porcine respiratory coronavirus (PRCV). Serum samples were obtained from pigs at various intervals from postinoculation day (PID) 0 through at least PID 22 to 40. Eleven-day-old pigs, seronegative for TGEV-neutralizing antibodies at the time of inoculation, were inoculated orally and nasally with either the virulent Miller (M5C) strain or the attenuated Purdue (P115) strain of TGEV, or with the ISU-1 strain of PRCV. Gastroenteritis was observed in 100% of the M5C-TGEV-inoculated pigs; but clinical signs of disease were not observed in either the P115-TGEV- or PRCV-inoculated pigs. Virus-neutralization (VN) antibody titer in sera was determined by use of a plaque-reduction assay. Blocking ELISA antibody titer for subsites A and D was determined from the serum dilution that produced 50% reduction in the absorbance values when it competed with biotinylated MAB 25C9 and 44C11, respectively. In sera from the inoculated pigs, the VN antibody titer began to increase by PID 7 and reached maximum by PID 15 to 16. For pigs inoculated with TGEV M5C, subsite A and subsite D blocking antibody titers in the serum paralleled the VN antibody titer, began to increase after PID 7, and reached maximum by PID 15 to 16.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of monoclonal antibodies to porcine group C rotaviruses cross-reactive with group A rotaviruses.

Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41-kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is recognized by certain MAbs in various serologic assays.

Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses.

A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.

Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV.

Five nonneutralizing monoclonal antibodies (MAb) generated to the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and specific for the S protein were characterized. Competition assays between purified and biotinylated MAb indicated that MAb 75B10 and 8G11 mapped near a new subsite, designated V and 2 MAb, 44C11 and 45A8, mapped to a previously designated subsite D. A fifth MAb mapped between subsites V and E. These MAb were tested with 3 previously characterized MAb to subsites A, E, and F in fixed-cell ELISA and cell culture immunofluorescent assays against 5 reference and 9 field strains of TGEV and 2 US strains (ISU-1 and ISU-3) of porcine respiratory coronavirus (PRCV). Subsites A, E, and F were conserved on all TGEV and PRCV strains examined. The 2 MAb to subsite V, 8G11 and 75B10, reacted only with the Miller TGEV strains (M5C, M6, and M60), except that 75B10 also recognized field strain U328. The MAb 11H8 did not react with 4 field strains or the Purdue strains of TGEV. The 2 MAb to subsite D reacted with all TGEV strains examined, but not with 2 US PRCV strains, 2 European PRCV strains, 1 feline infectious peritonitis virus strain, and 1 canine coronavirus strain. Because of this specificity for TGEV, but not PRCV, these latter 2 subsite D MAb may be useful for the development of competition ELISA to differentiate serologically between TGEV and PRCV infections in swine, similar to the currently used European subsite D MAb.

Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA.

A fixed-cell ELISA was developed using swine testicle (ST) cells infected with the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and purified biotinylated monoclonal antibodies (b-MAbs). Five of the b-MAbs were specific for the peplomer (E2), five reacted to the nucleocapsid (N), and one reacted to the E 1 protein of the Miller strain of TGEV. Protein A-Sepharose purification of MAbs yielded protein concentrations ranging from 0.40 to 3 mg per ml of ascites. Separate pools of N-MAbs and E 2-MAbs, and the E 1-MAb were used to monitor synthesis of TGE viral antigen in ST cells from 0 to 16 h post-infection at various multiplicities of infection (MOI). Epitopes of N proteins appeared sooner and at a lower MOI than those for the E 1 and E 2 proteins. The fixed-cell ELISA was also used to examine relative binding affinities of TGEV MAbs. Concentrations of b-MAbs producing a half-maximal signal ranged from 0.11 to 3.8 microgram/ml for E 2-MAbs, from 0.05 to 0.82 microgram/ml for N-MAbs, and 6 micrograms/ml for the E 1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E 2-MAbs (0.1 microgram/ml to 6.9 micrograms/ml) and one E 1-MAb (1.2 micrograms/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E 2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV.